Bulletin of Insectology 69 (1): 149-157, 2016 ISSN 1721-8861

Repellency of - and estragole-type fennel essential oils against stored grain pests: the different twins

1 2 3 1 2 Stefano BEDINI , Hind Houria BOUGHERRA , Guido FLAMINI , Francesca COSCI , Kamel BELHAMEL , 3 1 Roberta ASCRIZZI , Barbara CONTI 1Department of Agriculture, Food and Environment, University of Pisa, Italy 2Lab. of Organic Materials, Department of Process Engineering, Faculty of Technology, University of Bejaia, Algeria 3Department of Pharmacy, University of Pisa, Italy

Abstract

Aromatic plants essential oils (EOs) are promising alternatives to chemical insecticides and insect repellent for the post-harvest protection of crops. Fennel (Foeniculum vulgare Mill.) is a highly aromatic plant, cultivated worldwide of which several chemo- types can be distinguished on the basis of the relative content of its main compounds (E)-anethole and estragole. Fennel is well known for its pharmacological, antioxidant antimicrobial and acaricidal activities, and several studies showed its effectiveness as insecticidal and repellent against insects. In this study the repellency of the EOs extracted from two chemotypes, anethole- and estragole-type of F. vulgare fruits, against Rhyzopertha dominica (F.), Sitophilus zeamais Motschulsky and Tribolium confusum Jaqcquelin du Val, three of the major worldwide post-harvest grains insects was assessed by in vitro bioassays. Along with the EOs, we also tested the repellency of their major chemical components (E)-anethole, estragole, limonene and, fenchone and we evaluated the co-repellency effect of (E)-anethole and estragole. Finally, the repellence of the fennel EOs in the presence of maize was tested by a two-choice pitfall bioassay. (E)-anethole and estragole content in the anethole-type was 78.4 and 8.0%, respec- tively while in the estragole-type fennel EO anethole and estragole content was 0.9 and 85.5%, respectively. RD50 values showed that the estragole-type EO was the most effective repellent against the three insect species with values of 0.007, 0.051 and 1.124 mg cm-2 for R. dominica, T. confusum and S. zeamais, respectively. Consistently, relative median potency analysis showed that estragole was significantly more repellent to the three pest insect species than (E)-anethole. Interestingly, in the EOs, (E)-anethole and estragole showed a synergistic co-repellent effect. The strongest synergy was observed against R. dominica (CRC = 634.51). The repellency of fennel EOs also in the presence of maize was confirmed by a two-choice bioassay. Also in this case, the most overall effective EO resulted to be the estragole-type. The results highlight the importance of a chemical standardization based on the bioactivity of the fennel EOs and indicate the estragole-type fennel EO as suitable for the development of eco-friendly repel- lents for the post-harvest protection of grain crops.

Key words: post-harvest pest, anethole; estragole, insect repellency, synergy, fennel.

Introduction the potential variability of the EOs effectiveness. Fennel, Foeniculum vulgare Mill. (Apiaceae), is an Insects are major pests of stored food causing losses es- aromatic plant indigenous of the Mediterranean region timated around 20% of the annual world crop produc- well known for its pharmacological properties as carmi- tion (Sallam, 1999). Essential oils (EOs) of aromatic native, digestive, lactogogue and diuretetic (Manzoor et plants are effective natural products as contact and fu- al., 2012). The fennel, is largely distributed, especially migant insecticides and as repellents against stored food in dry soils, near the sea coast and on river banks, both pests (Isman, 2000; Nerio et al., 2009; Conti et al., as wild and cultivated plant (Napoli et al., 2010). The 2011; Athanassiou et al., 2013; Bougherra et al., 2015). species shows a large morphological and chemical di- Moreover, due to their high biodegradability and low versity. Some of the fennel genotypes that have been mammalian toxicity, EOs are regarded as very promis- entered in the European Pharmacopeia, are impossible ing tools for the formulation of rational low-toxic, eco- to be discriminated in relation to only morphological friendly food preservative (Isman, 2006). However, a data. However, several different chemotypes have been major difficulty for the implementation of aromatic identified on the basis of the EOs relative presence of plant EOs is that their bioactivity varies considerably the main compounds, the two enantiomers (E)-anethole depending to their chemical composition. EOs composi- and estragole (Krüger and Hammer, 1999). tion is reported to vary depending on a number of fac- Fennel EO has been already shown to have acaricidal tors such plant genetic structure (Telci et al., 2009), (Lee, 2004), antifungal (Singh et al., 2006), as well as phenological stage and site of origin of the plants antibacterial activity (Ruberto et al., 2000) and, re- (Shaaya and Kostyukovysky, 2006). Since the biologi- cently, its effectiveness as insecticidal and repellent cal activities of EOs depend on their chemical composi- against insects has been evaluated as well (Bertoli et al., tion, the chemical characterization coupled with the as- 2012). However, to our knowledge, no information is sessment of their biological properties is essential to available about the fennel EO effectiveness in relation standardize the chemical parameters that ensure the EOs to the chemical composition of its chemotypes. biological effects. Such standardization that has already The aim of the work was to assess the repellent activity been successfully introduced for the Australian Tea tree of different fennel EO chemotypes and of their main oil (Callander and James, 2012), could strongly reduce compounds against three of the major grain pests: the

lesser grain borer Rhyzopertha dominica (F.) (Bostrichi- Chemicals dae), the maize weevil Sitophilus zeamais Motschulsky (E)-anethole, estragole, limonene and, fenchone were (Dryophthoridae), and the confused flour beetle Tri- purchased from Sigma-Aldrich (Milano, Italy). Chemi- bolium confusum Jaqcquelin du Val (Tenebrionidae) in cals were of analytical standard grade. order to detect the relevant chemical parameters useful for a bioactivity based standardization of the fennel EOs. Insect cultures and rearing conditions R. dominica, S. zeamais and T. confusum were reared at the Department of Agriculture, Food and Environ- Materials and methods ment of the University of Pisa, since 2000. Insects were reared at room temperature (20-24 °C), 45-65% R.H., in Plant material the dark, in (20 × 27 × 11 cm) plastic boxes containing Estragole-type F. vulgare fruits were manually broken corn and wheat and covered by a nylon net al- harvested, at full ripeness, in Kabylia (Algeria) in the lowing air exchange. Insects homogeneous in age for summer 2012, dried in the shade, at room temperature the bioassays were obtained by removing the adults pre- (20-25 °C) until constant weight and stored in sent in the rearing boxes by sieving the grain and col- polyethylene bags in the dark at 4 °C. Seeds were then lecting the newly emerged insects (S. zeamais, 0-3 days transported in insulated polystyrene boxes on ice to the old; R. dominica and T. confusum, 0-1 day) the follow- laboratory of the University of Pisa for the EO ing day. extraction. Anethole-type F. vulgare fruits, were purchased from Repellence bioassays Biochimica srl (Arezzo, Italy). Area preference bioassays The repellence of the anethole- and estragole-type extraction and GC-MS analyses fennel EOs and of their main chemical compounds (E)- Fennel fruits were coarsely grounded in a mortar with anethole, estragole, limonene and, fenchone was as- a pestle and then subjected to hydro-distillation in a sessed by the area preference method described by Ta- modified Clevenger-type apparatus for 3 h (Guenter, pondjou et al. (2005). In detail, half filter paper disks (8 1949). The resulting essential oil was dried over anhy- cm ⌀) were treated with 500 µL of F. vulgare EO or drous sodium sulphate and stored in a glass vial at 4 °C pure chemical compounds as ethanolic solutions. Con- until use. trol half filter paper disks were treated with 500 µL of Gas chromatography (GC) analyses were carried out ethanol. Ethanol of the treated and control filter paper with an Hewlett Packard -5890 Series II instrument disks were evaporated under a fume hood. Each Petri equipped with HP-WAX and HP-5 capillary columns dish’s bottom (8 cm ⌀) was covered with half filter pa- (30 m × 0.25 mm, 0.25 µm film thickness), working with per treated with the EO or the chemical solution, while the following temperature program: 60 °C for 10 min, the other half, was covered with a control half filter pa- ramp of 3 °C min-1 up to 220 °C; injector and detector per disk. Repellency bioassays were performed with temperatures 250 °C; carrier gas helium (2 ml min-1); both F. vulgare EOs at doses ranging from 0.005 to detector dual FID; split ratio 1:30; injection of 0.5 µl 0.385 mg cm-2. Chemicals were tested at doses ranging (10% hexane solution). Components identification was from 0.01 to 0.74 mg cm-2. Twenty unsexed adults were carried out, for both columns, by comparing their reten- introduced in each Petri dish, and the lid was sealed tion times with those of pure authentic samples and by with Parafilm®. The Petri dishes were maintained in means of their linear retention index (LRI), relative to climatic chamber at 25 ± 1 °C, 65 ± 5% R.H., in the the series of n-hydrocarbons. Gas chromatography- dark, covered by black plastic pots. Five replicates were electron impact mass spectroscopy (GC-EIMS) analyses performed for each assay, and insects were used only were performed with a Varian CP-3800 gas chromato- once. The number of insects on the two half of the Petri graph, equipped with a HP-5 capillary column (30 m × dish was recorded after 24 h from the beginning of the 0.25 mm; coating thickness 0.25 µm) and a Varian Sat- test. The percent repellence (PR) of the EO and of each urn 2000 ion trap mass detector with the following ana- volatile compound was calculated by the formula: PR lytical conditions: injector and transfer line temperatures (%) = [(Nc − Nt) / (Nc + Nt)] × 100 where Nc is the 220 °C and 240 °C respectively; oven temperature pro- number of insects present in the control half paper and grammed from 60 °C to 240 °C at 3 °C min-1; carrier gas Nt the number of insects present in the treated one. helium at 1 ml min-1; injection of 0.2 µl (10% hexane solution); split ratio 1:30. Constituents identification was Two-choice pitfall bioassay based on the comparison of retention times with those of The potential protection of grains due to repellency of authentic samples, comparing their LRIs with the series the main volatile compounds of the fennel EOs, (E)- of n-hydrocarbons and using computer matching against anethole and estragole was evaluated against R. domin- commercial (Adams, 1995) and home-made library mass ica, S. zeamais, and T. confusum adults, using the two- spectra (built up from pure substances and components choice bioassay described by Germinara et al. (2007). of known oils and MS literature data (Adams, 1995). The two-choice pitfall bioassay for the presence of Moreover, molecular weights of all identified substances grains and because insects are never in direct contact were confirmed by gas chromatography-chemical ioniza- with the repellent substance, allows to test the repel- tion mass spectrometry (GC-CIMS), using methanol as lency of substances in conditions that are much more the chemical ionizing gas. close to a real situation respect to the area preference

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method. The bioassay was conducted in a steel arena Statistics and data analyses (32 cm ⌀ × 12 cm high) with two diametrically opposed Median repellent dose (RD50) was calculated by Log- holes (3 cm ⌀) located 3 cm from the sidewall, in the probit regression. Significant differences between RD50 bottom. 10 µl of EO or ethanol (control) were adsorbed values were determined by estimation of confidence in- onto a filter paper disk (1 cm ⌀) suspended at the centre tervals of the relative median potency (rmp). Differ- of each hole by a cotton thread taped to the lower sur- ences among RD50 values were judged to be statistically face of the arena. Glass flasks (500 ml) filled with 100 g significant when 1.0 was not found in the 95% confi- of maize grains (hybrid Eleonora, Pioneer Hi-Bred, It- dence interval of rmp. All the analyses were performed aly) were positioned under each hole, and the inside sur- by the SPSS 22.0 software (SPSS Inc., Chicago, IL, face of their necks were coated with paraffin oil to pre- USA). Co-repellency of (E)-anethole and estragole was vent insects, that have previously chosen, from return- evaluated using co-repellency coefficient (CRC) accord- ing to the arena. Preliminary trials allowed us to exclude ing to the co-toxicity coefficient calculation by Islam et any repellent or attractant effect of paraffin oil. The al. (2010) on the basis of the EOs components above floor of the arena was covered with filter paper to pro- 1% [(E)- anethole, estragole, limonene and, fenchone] vide an uniform surface and to facilitate insect move- using RD50 instead of LD50 values as follows: ments. Fifty insects, deprived of food for at least 4 - Repellency index (RI) of (E)-anethole = 100; hours, were placed under an inverted Petri dish (3 cm ⌀ - Repellency index (RI) of estragole = (RD50 of × 1.3 cm high) at the centre of the arena and allowed to (E)-anethole / RD50 of estragole) × 100; acclimate for 30 min. The arenas were covered with - Actual RI of EO = (RD50 of (E)-anethole / RD50 of steel lids and sealed with Parafilm® to prevent insects EO) × 100; from escaping and were left for 24 h in the dark at 25 ± - Theoretical RI of EO = RI of (E)-anethole × % of 1 °C and 65% R.H. Three replicates were performed for (E)-anethole in EO + RI of estragole × % of estragole each assay, and insects were used only once. The num- in EO + RI of limonene × % of limonene in EO + RI ber of insects in the flasks was recorded 24 h from the of fenchone × % of fenchone in EO; beginning of the test. The percent repellence (PR) of - CRC = (Actual RI of EO / Theoretical RI of EO) × 100. each volatile was then calculated after 24 h using the The effect was considered synergistic when CRC formula: PR (%) =[(Nc − Nt) / (Nc + Nt)] × 100 where > 120; indifferent when the CRC is > 80 to < 120 and Nc was the number of insects present in the control antagonistic when the CRC is < 80 (Islam et al., 2010). flask and Nt the number of insects present in the treated flask. Results

Table 1. Chemical composition (%) of the anethole- Essential oil extraction and GC-MS analyses (AEO) and estragole-type (EEO) F. vulgare essential EOs yield (w/w) from the anethole-type fennel was oils used in the repellency assays. 2.32%, whereas the yield from the estragole-type one

a was 2.25% dry weight. The EOs colour was pale yellow Constituents LRI AEO EEO and the smell long-lasting and very aromatic. α-pinene 941 0.6 0.2 In the anethole-type fennel essential oil (AEO), 24 sabinene 978 0.3 0.2 constituents were identified, accounting for 99.5% of myrcene 993 0.1 0.3 the whole oil. In the estragole-type fennel essential oil p-cymene 1028 0.4 0.4 (EEO), 19 constituents were identified, accounting for limonene 1032 6.2 7.5 99.9% of the whole oil (table 1). The principal chemical 1,8-cineole 1034 0.3 0.2 constituent of the AEO was (E)-anethole (78.4%) (Z)-β-ocimene 1042 0.2 0.5 whereas estragole (85.5%) was the main chemical in the γ-terpinene 1063 0.1 0.1 EEO (table 1). Other important volatiles were for both fenchone 1089 3.1 3.8 EOs limonene and fenchone (table 1). All other chemi- 1101 0.1 - cal constituents were below 1%. trans-p-mentha-2,8-dien-1-ol 1123 0.2 - The main chemical class was represented by phenyl- cis-limonene oxide 1136 0.1 - propanoids (86.5 and 86.4% for AEO and EEO, respec- cis-p-mentha-2,8-dien-1-ol 1139 0.1 - tively). The other classes were non-terpene derivatives, trans-limonene oxide 1142 0.1 - monoterpene hydrocarbons and oxygenated sesquiter- camphor 1145 0.2 0.1 penes (table 2). 4-terpineol 1179 - 0.1 estragol* 1197 8.0 85.5 Repellence bioassays carvone 1244 0.5 - Area preference bioassays p-anisaldehyde 1255 0.5 - Both fennel EOs showed a good repellent activity (E)-anethole* 1285 78.4 0.9 against the three insects R. dominica, S. zeamais and dill 1623 - 0.1 T. confusum. AEO RD50 ranged from 0.056 to 0.1 mg Total identified 99.5 99.9 -2 cm against R. dominica and T. confusum, respectively a Chemical constituents ≥ 0.1%; LRI, linear retention while, EEO RD50 values ranged from 0.007 to 0.051 mg index on DB-5 column; * chemicals tested for insect cm-2 against R. dominica and T. confusum, respectively pests repellency. (table 3).

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Table 2. Principal chemical classes (%) in the anethole- significant differences of sensitiveness to (E)-anethole (AEO) and estragole-type (EEO) F. vulgare essential were found among species (table 5). The comparison of oils used in the repellency assays. the EOs components bioactivity by rmp analyses showed that estragole was significantly more active than Chemical classes AEO EEO (E)-anethole against S. zeamais and R. dominica with Monoterpene hydrocarbons 9.2 7.9 Log rmp values (estragole vs (E)-anethole) ranging from Non-terpene derivatives - 0.5 −1.206 to −0.146 for R. dominica and T. confusum, Oxygenated monoterpenes 4.2 4.7 respectively (figure 1). CRC values showed an overall 86.5 86.4 synergistic effect (CRC values > 120) of (E)-anethole Not identified 0.1 0.5 and estragole against the three insects species with the

exception of estragole-type EO against S. zeamais and Consistently with fennel EOs, their main components (E)-anethole-type EO against T. confusum whose effects (E)-anethole and estragole, exerted a clear repellent ac- are to be considered as addictive with CRC values of 118.27 and 108.13, respectively. The strongest tivity against insect pests. The RD50 of (E)-anethole ranged from 0.612 to 0.094 mg cm-2 for R. dominica synergistic effect was observed against R. dominica with CRC values of 537.71 and 634.51 for the anethole- and and T. confusum, respectively, while the RD50 of the es- tragole ranged from 0.038 to 0.060 mg cm-2 for R. do- estragole-type, respectively (table 3). minica and S. zeamais, respectively (table 4). A strong repellency was also observed for fenchone and limo- Two-choice pitfall bioassay nene against T. confusum and R. dominica (table 4). The effect of the fennel EOs main components in the Interestingly, rmp analyses showed a significant presence of maize was tested by a two-choice pitfall bio- different sensitiveness among species to estragole. In assay. In line with the area preference bioassay, the most detail, rmp analyses indicate that the most sensitive overall effective EO resulted to be the estragole-type species to estragole was R. dominica while the less with repellency values of 71.9 ± 24.48, 28.5 ± 9.95 and sensitive was S. zeamais (table 5). On the contrary, no 58.1 ± 7.66% for R. dominica, S. zeamais and T. con-

Table 3. Repellency of anethole- (AEO) and estragole-type (EEO) F. vulgare essential oils (EOs) against adults of R. dominica, S. zeamais and T. confusum.

a b c 2 d EOs CRC RD50 95% CI Slope ± SE Intercept ± SE χ (df) AEO 537.71 0.056 0.019-0.080 1.353 ± 0.460 1.353 ± 0.439 0.23 (1) R. dominica EEO 634.51 0.007 0.003-0.014 0.651 ± 0.125 1.386 ± 0.227 1.49 (2) AEO 168.09 0.176 0.156-0.198 4.672 ± 0.662 3.447 ± 0.493 0.38 (1) S. zeamais EEO 118.27 0.124 0.078-0.265 0.896 ± 0.217 0.798 ± 0.258 0.13 (2) AEO 108.13 0.100 0.082-0.122 2.681 ± 0.470 2.640 ± 0.481 0.01 (1) T. confusum EEO 136.68 0.051 0.018-0.075 1.245 ± 0.343 1.589 ± 0.338 2.20 (2) a Co-repellency coefficient (CRC). CRC< 80 is considered as antagonistic, 80 < CRC < 120 as additive, CRC > 120 as synergistic; b Concentration of the extract that repels 50% of the exposed insect. Data are expressed as mg cm-2; c Confidence Interval; d Chi-square (df) degrees of freedom; Values in bold indicate P > 0.05.

Table 4. Repellency of (E)-anethole and estragole, limonene and fenchone against adults of S. zeamais, T. confusum and R. dominica.

a b 2 c Repellent Pest target RD50 95% CI Slope ± SE Intercept ± SE χ (df) R. dominica 0.612 0.232-34.890 0.542 ± 0.187 0.116 ± 0.210 0.83 (2) (E)-anethole S. zeamais 0.271 0.163-0.682 0.838 ± 0.201 0.47 ± 0.200 2.71 (2) T. confusum 0.094 0.072-0.118 2.247 ± 0.329 2.311 ± 0.337 1.16 (1) R. dominica 0.038 0.000-0.849 0.580 ± 0.252 0.826 ± 0.299 0.01 (1) Estragole S. zeamais 0.126 0.035-0.234 0.869 ± 0.342 0.782 ± 0.294 1.51 (1) T. confusum 0.060 0.040-0.084 1.315 ± 0.194 1.607 ± 0.230 0.09 (2) R. dominica 0.099 0.058-0.176 0.904 ± 0.270 0.906 ± 0.289 0.33 (2) Limonene S. zeamais 0.213 0.148-0.654 1.414 ± 0.458 0.950 ± 0.433 1.71 (1) T. confusum 0.409 0.165-967.012 1.007 ± 0.415 0.391 ± 0.504 0.54 (2) R. dominica 0.310 0.128-9.698 0.825 ± 0.269 0.419 ± 0.317 1.78 (1) Fenchone S. zeamais 0.417 0.096->1000 0.428 ± 0.200 0.163 ± 0.311 0.02 (1) T. confusum 0.062 0.043-0.095 1.347 ± 0.272 1.615 ± 0.358 1.36 (1) a Concentration of repellent that repel 50% of the exposed insects. Data are expressed as mg cm-2; b Confidence In- terval; c Chi-square (df) degrees of freedom; Values in bold indicate P > 0.05.

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Table 5. Relative susceptibilities of adults of R. domin- and that there were no interaction between species and ica, S. zeamais and T. confusum to (E)-anethole and EO (F2, 18 = 8.419, P = 0.005). Besides, we observed a estragole. different number of insects that did not choice either of the two treated and non-treated grain chambers. The Component T. confusum R. dominica non-choosing individuals in the tests with the estragole S. zeamais 0.325 1.139 (E)-anethole were 38.0 ± 3.1, 11.0 ± 1.2 and 5.0 ± 2.6 for R. domin- R. dominica 0.285 ica, S. zeamais and T. confusum, respectively, while S. zeamais 0.420a 0.393 Estragole non-choosing individuals of (E)-anethole were 39.0 ± R. dominica 1.069 3.5, 12.3 ± 2.4, and 10.7 ± 1.2 for R. dominica, S. zea- a relative median potency (rmp) analyses values of the mais and T. confusum, respectively. Significant differ- comparisons: T. confusum vs S. zeamais and R. domin- ences of the number of non-choosing individuals were ica; R. dominica vs S. zeamais. Values < 1 indicates found, as a function of species (F1, 18 = 64.926, P < more susceptibility; Values > 1 indicates less suscepti- 0.001) but not of the EO (F2, 18 = 44.290, P = 0.093) bility. In bold the significant values (95% CI ≠ 1). and there were no interaction between species and EO (F2, 18 = 1.805, P = 0.206). fusum, respectively, while the repellency of (E)-anethole was 48.9 ± 9.9, −20.5 ± 3.1 and 37.7 ± 10.6% for Discussion R. dominica, S. zeamais and T. confusum, respectively (figure 2). The negative value of (E)-anethole indicates Chemical analyses showed considerable quantitative that, at the doses tested, it had an attractive activity for rather than qualitative differences in the chemical com- S. zeamais (figure 2). Consistently with the area prefer- position of the two EOs. In particular, EEO and AEO ence method, the two choice tests confirmed R. domin- exhibited a very different proportion of the two isomers ica as the most susceptible species to the repellency of estragole and (E)-anethole. While (E)-anethole was the fennel EOs and S. zeamais the less one. Two-ways main component of the AEO, the main component of ANOVA showed significant differences between the the EEO was estragole. Overall, yields and chemical two EOs chemotypes, as a function of species (F2, 18 = composition of F. vulgare EOs match with previous 4402.542, P < 0.001) the EO (F1, 18 = 34.834, P < 0.001) studies on fennel (Napoli et al., 2010; Diao et al., 2014;

0.5

0

-0.5

-1

-1.5 Log rmp Log -2

-2.5

-3

-3.5

Figure 1. Relative median potency (rmp) comparison between the repellency of (E)-anethole and estragole against the insect pests assessed by area preference bioassay. Values < 0 indicates a stronger repellency of estragole respect to anethole. Bars crossing the zero line indicate that the difference of effectiveness is not statistically significant. R. dominica, white; S. zeamais, grey; T. confusum, black.

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100

80

60

40

20 % of repellency %

0 R. dominica S. zeamais T. confusum

-20

-40

Figure 2. Repellency of anethole- and estragole-type fennel essential oils against the insect pests assessed by two- choice pitfall bioassay. Anethole-type, light gray; estragole-type, dark gray. Bars indicate standard errors.

Özcan et al., 2006) confirming a strong difference in the respectively) (Bougherra et al., 2015). chemical composition of fennel EOs chemotypes (Ba- Even if both repellent against the insects, significant doc et al., 1998). differences in the effectiveness between the two EOs Although at different intensity, both the fennel chemo- chemotypes were observed. Such differences can be ex- types AEO and EEO, were able to exert a repellent ac- plained on the basis of the different effectiveness ob- tivity against the three pest insect species. To that re- served between the two enantiomers (E)-anethole and gard, our data are overall in accordance with previous estragole. In fact, rmp analysis showed that estragole is studies showing a repellent effect of fennel EO against more effective as insect repellent than (E)-anethole par- food-stuff pests. Cosimi et al. (2009) found that an ticularly against R. dominica (rmp value = 0.062). Ac- anethole-rich fennel EO exerted a moderate level of re- cordingly to our results, Kim and Lee (2014) found that pellency against S. zeamais, lower than the repellency of estragole was the most active compound also of bergamot and lavandin. Similarly, insecticidal activity and orange EOs, as adulticidal, against S. zeamais and of an anethole-rich fennel EO was also observed against the red flour beetle Tribolium castaneum (Herbst). Simi- the granary weevil S. granarius L. by Zoubiri and larly, estragole was found to be more effective as adulti- Baaliouamer (2011) and by Bertoli et al. (2012) who, cidal agent than (E)-anethole against Sitophilus oryzae comparing six aromatic plant EOs, observed that an es- (L.), Callosobruchus chinensis (L.), and Lasioderma tragole rich fennel essential oil showed the highest con- serricorne (F.) (Kim and Ahn, 2001). Similar differ- tact toxicity rates, at any concentrations. ences between enantiomers were previously evidenced In comparison with the repellent activity of other aro- also for two enantiomeric forms of limonene, which al- matic plants essential oils, both the fennel EOs resulted though similar in the LC50, showed significant differ- in line with the effectiveness of Algerian Pistacia len- ences in their repellent activity against the Asian tiger tiscus L. and Laurus nobilis L. EOs (RD50 = 0.037 and mosquito Aedes albopictus (Skuse) (Diptera Culicidae) 0.033 µL cm-2, for P. lentiscus and L. nobilis, respec- (Giatropoulos et al., 2012). Besides (E)-anethole and tively) against R. dominica (Mediouni Ben Jemâa et al. estragole, in our experiment, we observed a strong re- 2012; Bougherra et al., 2015) but lower than the one pellent activity also by limonene and fenchone against reported for Algerian P. lentiscus EOs against S. zea- R. dominica and T. confusum, respectively. Consis- -2 mais and T. confusum (RD50 = 0.010 and 0.025 µL cm , tently, a similar repellent activity of limonene against

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R. dominica was previously observed by Bedini et al. with the best result obtained with estragole-type fennel (2015) who also observed that limonene was the most EO against R. dominica. Besides, we observed a differ- active component of hops EO against S. granarius. ent percentage of individuals among species that did not Our results also showed a different susceptibility of choice any of the two flasks containing maize. A high the three insect species to fennel EOs and their major percentage of non-choosing individuals for R. dominica chemical components. Among the three species tested, was already observed and could be due to characteristic R. dominica was the overall most susceptible specie behavior of this species (Bougherra et al., 2015). while S. zeamais the less sensitive one. These results are in contrast with the findings of Bougherra et al. (2015) who observed a higher susceptibility of S. zeamais re- Conclusions spect to R. dominica to the P. lentiscus EO, but they are consistent with the bioactivity of spent hops (Humulus Overall, this experiment contributes to the knowledge lupulus L.) EO (Bedini et al., 2015) that was about 24 about the chemical composition and bioactivity of the time higher against R. dominica than against S. fennel EOs chemotypes. Even if, in a real world sce- granarius. These findings indicate that the efficacy of nario, repellence alone would probably be insufficient to EOs as repellent depends not only on their chemical provide complete protection to stored grains, by lower- composition but also on the target species that may be ing the insect infestation level, it could highly facilitate differently susceptible to the different chemical com- the action of other parallel control methods. In this re- pounds. Accordingly, in this experiment, the rmp analy- gard, estragole-type fennel EO also because of the syn- ses showed that estragole was not only the overall most ergistic effect between its main components, resulted effective compound of fennel EOs but also the com- significantly more effective that anethole-type fennel pound whose activity is more clearly dependent on the EO. Thus, the standardization of fennel EOs by es- species. tragole content could be a useful step toward the devel- In addition we observed that the effectiveness of the opment of reliable low-toxic eco-friendly repellents able fennel EOs against R. dominica was much higher than to reduce the post-harvest grain losses caused by insect the one of (E)-anethole and estragole alone. Interest- pests. ingly, CRC calculation showed a strong synergistic ef- fect of (E)-anethole and estragole against R. dominica. Such synergistic effect could explain the stronger effec- Acknowledgements tiveness of the fennel EOs against R. dominica respect to the other two species. To our knowledge, this is the We would like to thank Adjaoud Abdenour (Faculté des first experiment evaluating the contribution of the syn- Sciences de la Nature et de la Vie, University of Bejaia, ergistic effect of essential oil components in the insect Algeria) for the confirmation of the species identifica- repellency. However, several previous experiments on tion of the collected plants and Paolo Giannotti for the insect toxicity are consistent with our results showing skilled assistance during the set-up of the experiment. that the combined effect of bioactive substances on in- sects is synergistic, addictive or antagonistic depending on the substances and on the insect species (Sun and References Johnson, 1960; Jiang et al., 2009; Pavela, 2014). Simi- ADAMS R. P., 1995.- Identification of essential oil components larly, Savelev et al. (2003) found a complex interaction by Gas-Chromatography-Mass-Spectrometry.- Allured Pub. among the constituents of Salvia lavandulaefolia Vahl Co., Carol Stream, IL, USA. EO in the inhibition of acetylcholinesterase with both ATHANASSIOU C. G., KAVALLIERATOS N. G., EVERGETIS E., synergistic and antagonistic effects between the compo- KATSOULA A. M., HAROUTOUNIAN S. A. 2013.- Insecticidal nent terpenes, while Hummelbrunner and Isman (2001) efficacy of silica gel with Juniperus oxycedrus ssp. oxy- observed a synergistic effect of (E)-anethole with thy- cedrus (Pinales: Cupressaceae) essential oil against Sitophi- mol, citronellal, and α-terpineol, in acute toxicity and lus oryzae (Coleoptera: Curculionidae) and Tribolium con- feeding deterrence on the tobacco cutworm, Spodoptera fusum (Coleoptera: Tenebrionidae).- Journal of Economic litura (F.) (Lepidoptera Noctuidae). Entomology, 106: 1902-1910. 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TAPONDJOU A., ADLER C., FONTEM D., BOUDA H., REICHMUTH Authors’ addresses: Barbara CONTI (corresponding au- C., 2005.- Bioactivities of cymol and essential oils of Cu- thor, [email protected]), Stefano BEDINI, Francesca pressus sempervirens and Eucalyptus saligna against Sito- COSCI, Department of Agriculture, Food and Environment, philus zeamais Motschulsky and Tribolium confusum du University of Pisa, via del Borghetto 80, 56124 Pisa, Italy; Val.- Journal of Stored Product and Research, 41: 91-102. Hind Houria BOUGHERRA, Kamel BELHAMEL, Laboratory of TELCI I., DEMIRTAS I., SAHIN A., 2009.- Variation in plant Organic Materials, Department of Process Engineering, Fac- properties and essential oil composition of sweet fennel ulty of Technology, University of Bejaia, DZ 06000, Algeria: (Foeniculum vulgare Mill.) fruits during stages of maturity.- Guido FLAMINI, Roberta ASCRIZZI, Department of Pharmacy, Industrial Crops and Products, 30: 126-130. University of Pisa, via Bonanno 33, 56126 Pisa, Italy. ZOUBIRI S., BAALIOUAMER A., 2011.- Chemical composition and insecticidal properties of some aromatic herbs essential oils from Algeria.- Food Chemistry, 129: 179-182. Received December 17, 2015. Accepted March 21, 2016.

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